Cell death is essential for many physiological processes, and its deregulation characterizes numerous human diseases. Thus, in-depth investigation of cell death and its mechanism has tremendous implications for the development of novel therapeutic strategies. This is especially true for eosinophils, whose extended survival and activation or necrotic cell death with release of toxic granule proteins lead to tissue inflammation in eosinophil-associated diseases. Classically, cell death was divided dichotomously into apoptotic and necrotic; however, recent studies have suggested the existence of a continuum of cell death phenotypes, as well as novel, distinct cell death processes such as regulated necrosis and, in certain situations, autophagy. Importantly, these subtypes are differentially regulated by specific biochemical cascades; thus, the correct identification of cell death phenotype may have important therapeutic implications, as cells may be targetable by regimens that induce or inhibit a specific mode of cell death. Regulated necrosis commonly occurs in situations in which cells receive a cell death signal but apoptosis is inhibited (e.g. Fas ligation concurrent with caspase inhibition). Similarly, we observed the paradoxical enhancement of cell death in eosinophils simultaneously treated with survival factors (e.g. IL-5, acidity) and cell death-inducing agents (anti-Fas, anti-Siglec-8). Moreover, th mode of cell death was distinct; anti-Siglec-8 induced caspase-dependent apoptosis whereas anti-Siglec-8 in IL-5-treated eosinophils caused caspase- independent cell death. Conceptually, these findings are consistent with the notion that in the tissue microenvironment, eosinophils are exposed to multiple signals simultaneously, including pro-survival and pro- cell death signals. Indeed, in tissue samples collected from patients with eosinophilic inflammatory disease, a large portion of eosinophils display ultrastructural characteristics of cytolysis or necrosis. However, the spectrum of cell death phenotypes induced in eosinophils and the biochemical mechanisms leading to these modes of cell death are not known. The studies proposed in this grant application aim to address this gap in knowledge and to serve as a platform for the eventual translation of this knowledge to clinical settings. Our central hypothesis is that eosinophils undergo regulated necrosis, a targetable process, which has important pathophysiological implications in eosinophil-associated disease. We propose two specific aims to test this hypothesis: 1) to define the spectrum of human eosinophil cell death phenotypes, and 2) to determine the pathophysiological consequences of regulated necrosis of eosinophils. We will use innovative approaches in primary human eosinophils, biopsies from patients with eosinophilic disease, and animal models. Our studies will provide proof-of-concept that regulated necrosis occurs in eosinophils (aim 1) and that it is significant in disease (aim 2), which will provide critical preliminary data for a mechanistic R01-level grant application.